Comparison of neurovirulent and attenuated poliovirus genomes has implicated the 5' noncoding region of the viral RNA in a rate limiting step in viral growth and neurovirulence. The poliovirus 5' noncoding region presumably contains signals important for the translation and amplification of the viral RNA genome. The availability of an infectious cDNA clone of the viral genome and the discovery that in vitro synthesized viral RNA is infectious when introduced into mammalian cells, allow the application of genetics to study this RNA virus. The aim of this proposal is to introduce defined mutations into the 5' noncoding region by site- directed mutagenesis of the cDNA and to study their effects on the translation and amplification of the viral RNA genome. Specifically, we will analyze wild type and mutant RNA molecules in in vitro translation and replication systems. Hybrid genes containing the 5' noncoding region of wild type or altered viral genomes and the coding region of a test gene will be constructed and the translatability of the hybrid mRNAs will be tested in cell lines harbouring the hybrid genes stably integrated in the chromosome. In addition, we will search for trans-acting factors which might interact with the 5' noncoding region. These studies will be complemented by the structural analysis of wild type and mutant 5' noncoding regions employing RNA-conformation specific chemical probes and reverse transcriptase. The recent sequencing of other picornavirus RNA genomes has revealed great homology in the 5' noncoding region. Thus, knowledge of the structure and function of this region will increase our understanding of the neurovirulent behaviour of other medically important RNA viruses such as Theiler's virus, a neurotropic mouse RNA virus, which is studied extensively in the laboratories of Drs. Murray and Rotbart at the University of Colorado Health Sciences Center.